Epithelial ovarian cancer (EOC) remains the leading cause of death by gynecological malignancy in the United States. Thus, there is an urgent need for novel EOC therapeutics. Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that silences gene expression by generating tri-methylation on lysine 27 residue of histone H3 (H3K27Me3). We recently published that EZH2 is frequently overexpressed in primary human EOCs. However, the mechanism underlying EZH2 upregulation in EOC is poorly understood. We have evidence to suggest that the CCAAT binding transcription factor NF-Y plays a critical role in regulating EZH2 expression in EOC cells. Significantly, we have showed that EZH2 expression regulates the proliferation, apoptosis and invasion of EOC cells. The objective of this proposal is to investigate the molecular basis whereby EZH2 promotes the malignant behaviors of EOC cells. We performed whole-genome chromatin immunoprecipitation (ChIP) followed by next generation sequencing analysis and gene expression profiling. Integrative analysis revealed a list of 60 direct EZH2 target genes whose expression was upregulated by EZH2 knockdown in EOC cells. To prioritize the identified genes for validation, we examined their expression in the Cancer Genome Atlas EOC database and discovered that 3 of the 60 genes are downregulated >2-fold with concurrent EZH2 upregulation in >75% of EOC cases. Interestingly, 3 prioritized genes are implicated in regulating cell growth, invasion, apoptosis and/or cancer stem cells. We validated the binding of EZH2/H3K27Me3 to their promoters by ChIP and confirmed their upregulation in EZH2 knockdown cells. Our central hypothesis is that EZH2 regulates malignant behaviors of EOC cells by epigenetically silencing the target genes we have identified. Our long-term goal is to target EZH2 for developing novel EOC therapeutics. Here we showed that two novel specific inhibitors of EZH2 methyltransferase activity suppress the proliferation and invasion of EOC cells. Three specific aims are proposed. Aim 1: To investigate the role of the CCAAT binding transcription factor NF-Y in regulating EZH2 expression in human EOC cells; Aim 2: To investigate the expression pattern and function of the validated EZH2 target genes in human ovarian tumors; and Aim 3: To investigate whether the effects observed in EOC cells treated with inhibitors of EZH2 methyltransferase activity are mediated by the validated EZH2 target genes. The research proposed is innovative because it will not only elucidate a novel molecular mechanism underlying EZH2 upregulation, but also provide new mechanistic insights into how EZH2 contributes to malignant behaviors of EOC. In addition, the proposed studies will reveal the mechanisms of action of two novel inhibitors of EZH2 methyltransferase. These studies are significant because it has potential to establish EZH2 as a novel target for developing EOC therapeutics. In addition, the mechanistic insights gained from the current studies have broad implications for many different types of cancers as well.